Amplification of c-myc, K-sam, and c-met in gastric cancers: detection by fluorescence in situ hybridization

Gene amplifications of c-myc, K-sam, and c-met were examined in cancer nuclei isolated from 154 primary gastric adenocarcinomas by fluorescence in situ hybridization (FISH) using cosmid probes for 8q24 (c-myc locus) and 7q31 (c-met), as well as a DNA probe for K-sam synthesized by PCR. The results were compared with those of Southern blot analysis. Dual-color FISH using gene locus and chromosome-specific probes detected gene amplifications of c-myc in 24 tumors (15.5%), c-met in 6 tumors (3.9%), and K-sam in 3 tumors (2.9%). The six tumors with c-myc amplification had also been found to have amplified c-erbB-2 in our previous study, and coamplification of c-myc and c-met was found in two other tumors. This technique also differentiated the amplified genes on the homogeneous staining region (HSR) and on double minute chromosomes (DMs) in metaphase spreads and interphase nuclei of cell lines established from poorly differentiated adenocarcinomas, KATO III, SNU 16, and HSC 39. Examination of FISH images of these cell lines suggested that the high-level amplifications of c-myc found in primary tumors occurred mainly on DM in four tumors and on HSR in one, and those of K-sam occured on DM in two tumors and on HSR in one. No high-level amplification of c-met was found. These high-level amplifications were also detected in formalin-fixed, paraffin-embedded tissues from primary gastric tumors and metastatic lymph nodes, in some of which heterogeneity of gene amplification was demonstrated within the same tumor. We conclude that FISH is an important tool for examining the proto-oncogene aberrations in intact cells in solid tumors.     

Molecular cloning of the actin gene from yeast Saccharomyces cerevisiae

Two overlapping DNA fragments from yeast Saccharomyces cerevisiae containing the actin gene have been inserted into pBR322 and cloned in E.coli. Clones were identified by hybridization to complementary RNA from a plasmid containing a copy of Dictyostelium actin mRNA. One recombinant plasmid obtained (pYA102) contains a 3.93-kb Hindlll fragment, the other (pYA208) a 5.1-kb Pstl fragment, both share a common 2.2-kb fragment harboring part of the actin gene. Cloned yeast actin DNA was identified by R-loop formation and translation of the hybridized actin mRNA and by DNA sequence analysis. Cytoplasmic actin mRNA has been estimated to be about 1250 nucleotides long. There is only one type of the actin gene in S.cerevisiae.     

The penicillin gene cluster is amplified in tandem repeats linked by conserved hexanucleotide sequences

The penicillin biosynthetic genes (pcbAB, pcbC, penDE) of Penicillium chrysogenum AS-P-78 were located in a 106.5-kb DNA region that is amplified in tandem repeats (five or six copies) linked by conserved TTTACA sequences. The wild-type strains P. chrysogenum NRRL 1951 and Penicillium notatum ATCC 9478 (Fleming's isolate) contain a single copy of the 106.5-kb region. This region was bordered by the same TTTACA hexanucleotide found between tandem repeats in strain AS-P-78. A penicillin overproducer strain, P. chrysogenum E1, contains a large number of copies in tandem of a 57.9-kb DNA fragment, linked by the same hexanucleotide or its reverse complementary TGTAAA sequence. The deletion mutant P. chrysogenum npe10 showed a deletion of 57.9 kb that corresponds exactly to the DNA fragment that is amplified in E1. The conserved hexanucleotide sequence was reconstituted at the deletion site. The amplification has occurred within a single chromosome (chromosome I). The tandem reiteration and deletion appear to arise by mutation-induced site-specific recombination at the conserved hexanucleotide sequences.     

Identification of a novel tandemly repeated sequence present in an intron of the glucose phosphate isomerase (GPI) gene in mouse and man

Glucose phosphate isomerase (GPI, glucose 6-phosphate ketol-isomerase, EC 5.3.1.9) is a housekeeping gene expressed in all tissues and organisms that utilize glycolysis and gluconeogenesis. Deficiency in humans leads to a rare form of nonspherocytic hemolytic anemia. We have isolated a 3.2-kb mouse cDNA containing glucose phosphate isomerase coding sequence and a 2.1-kb intronic sequence and a large proportion of the human gene (approaching 55 kb) in four phage lambda recombinants. A 4-kb intronic fragment from the human gene showing homology to the mouse intronic sequence has been isolated and sequenced. The fragment contains approximately 1.5 kb of sequence that is composed of 30 repeat units of a novel 50-bp tandemly repeated units. The mouse intronic sequence contains 18 similar units. The human consensus sequence differs from the mouse consensus sequence at only 7 positions out of 50 (positions 16, 26, 27, 42, 43, 47, and 48). A probe containing the repeat element detects polymorphisms, specific to glucose phosphate isomerase, in human DNA. The repeat element does not appear to be present at any other loci in human DNA. The conservation of this intronic repeat element extends to pig and Chinese hamster.     

Quantitation of HER-2/neu and c-myc gene amplification in breast carcinoma using fluorescence in situ hybridization

HER-2/neu and c-myc amplification or overexpression have been reported to be associated with poor prognosis in breast carcinoma. The prognostic significance, however, remains somewhat controversial, partly because of discrepancies among different methodologies used for detection of the oncogene amplification or overexpression. Fluorescence in situ hybridization (FISH) has recently been shown to be a useful technique for analyzing genetic alterations in interphase nuclei in various tumors. In this study, FISH was used to quantitate HER-2/ neu and c-myc gene amplification in touch preparations of frozen tissue from 100 node-negative breast carcinomas. HER-2/neu amplification was found to be associated with an abnormal DNA index (P < .001) and tumor size (P < .04). Amplification of c-myc was associated with S phase (P < .0003), abnormal DNA index (P < .003), and a negative estrogen receptor status (P < .01). The coamplification of both oncogenes was strongly associated with an abnormal DNA index (P < .0001) and with tumor size (P < .009). The use of FISH for detection of HER-2/neu gene amplification was 92% concordant with immunocytochemistry (ICC) used for detection of overexpression of HER-2/neu protein. Fifteen of the 100 cases were both amplified for HER-2/neu by FISH and positive by ICC analysis. Seven cases without HER-2/neu gene amplification demonstrated HER-2/neu protein overexpression by ICC. One HER-2/neu-amplified case was negative by ICC. Repeat analysis of a subset of cases showed FISH to be a more reproducible method than ICC in the analysis of HER-2/neu in touch preparations of breast carcinoma. FISH is a rapid and reproducible method that allows the accurate measurement of the level of oncogene amplification within interphase nuclei. The use of FISH should provide a more accurate assessment of the prognostic significance of oncogene amplification in breast carcinoma.     

Amplification of the c-myc gene in human hepatocellular carcinoma: biologic significance

To elucidate the prevalence and biologic significance of the c-myc gene in human hepatocellular carcinoma (HCC), DNA samples were taken from the paired tumorous and nontumorous tissues of 77 cases of resected primary HCC and were analyzed by Southern blot hybridization. We demonstrated modest, but significant c-myc amplification (group A) in 28 (36.4%) of the cases: 1.6- to 2.0-fold in 18, 2.1- to 3.0-fold in four, and > 3.0-fold in six. Compared to HCC without c-myc amplification (group B), group A HCC occurred more often in patients < 50 years old (54.5% vs 29.1%, p < 0.02) with serum alpha-fetoprotein (AFP) levels > 320 ng/mL (61.1% vs 14.6%, p < 0.00002). Group A HCC occurred more frequently in patients with hepatitis B virus infection than in those with hepatitis C virus infection (p < 0.03). Group A HCC was more likely to be poorly differentiated (44.8% vs 10.5%, p < 0.004) and associated with intrahepatic portal vein spread (57.1% vs 28.6%, p < 0.02). The c-myc amplification did not correlate with sex or tumor size. For small HCC, group A had a worse one-year survival rate than group B (72.2% vs 90.9%, p < 0.04). These findings suggest that c-myc amplification is not an uncommon event in human hepatocarcinogenesis, occurs more frequently in young patients who have an elevated serum AFP level or HBV infection, and is related to the biologic behavior of HCC.     

An mpb-64 flanking sequence specific for Mycobacterium bovis

A clone carrying a plasmid with the mpb-64 gene and 3' flanking sequences (plasmid pMBA122) was detected during the screening of a Mycobacterium bovis genomic library with sera from infected cattle. When the pMBA122 insert was used as a probe in Southern blots against PvuII-digested mycobacterial DNA, it distinguished the different M. tuberculosis complex species. This probe hybridized with a 7-kb band in M. tuberculosis, a 5-kb band in M. bovis and a 3-kb band in M. tuberculosis complex strains from wild seals. Smal genomic digestions enabled us to locate this polymorphic region downstream of the mpb-64 gene. In order to clone this particular region, we designed a pair of PCR primers. Unexpectedly, these primers amplified only M. bovis DNA; no amplification was seen in M. tuberculosis DNA. When the annealing temperature was lowered from 70 to 55 degrees C, an amplification product of the same size was obtained with M. tuberculosis. This product was cloned and sequenced, and showed partial homology to the M. bovis amplified fragment. Therefore, this region comprises M. bovis sequences with a lower homology with M. tuberculosis than other compared sequences. This suggests that a more precise differentiation method at the species level for the M. tuberculosis complex could be achieved using PCR directed to this region.     

Genetic analysis of type 5 capsular polysaccharide expression by Staphylococcus aureus

Capsules are produced by over 90% of Staphylococcus aureus strains, and approximately 25% of clinical isolates express type 5 capsular polysaccharide (CP5). We mutagenized the type 5 strain Reynolds with Tn918 to target genes involved in CP5 expression. From a capsule-deficient mutant, we cloned into a cosmid vector an approximately 26-kb EcoRI fragment containing the transposon insertion. In the absence of tetracycline selection, Tn918 was spontaneously excised, thereby resulting in a plasmid containing 9.4 kb of S. aureus DNA flanking the Tn918 insertion site. The 9.4-kb DNA fragment was used to screen a cosmid library prepared from the wild-type strain. Positive colonies were identified by colony hybridization, and a restriction map of one clone (pJCL19 with an approximately 34-kb insert) carrying the putative capsule gene region was constructed. Fragments of pJCL19 were used to probe genomic DNA digests from S. aureus strains of different capsular serotypes. Fragments on the ends of the cloned DNA hybridized to fragments of similar sizes in most of the strains examined. Blots hybridized to two fragments flanking the central region of the cloned DNA showed restriction fragment length polymorphism. A centrally located DNA fragment hybridized only to DNA from capsular types 2, 4, and 5. DNA from pJCL19 was subcloned to a shuttle vector for complementation studies. A 6.2-kb EcoRI-ClaI fragment complemented CP5 expression in a capsule-negative mutant derived by mutagenesis with ethyl methanesulfonate. These experiments provide the necessary groundwork for identifying genes involved in CP5 expression by S. aureus.     

Characterization of human endotoxin lipopolysaccharide receptor CD14 expression in transgenic mice

CD14 is a major receptor for the bacterial endotoxin LPS. Since CD14 is specifically and highly expressed on the surface of monocytic cells, it has been used as a monocyte/macrophage differentiation marker. To identify elements that are critical for the direction of the tissue-specific expression of CD14, an 80-kb genomic DNA fragment containing the coding region of the CD14 gene, as well as a considerable amount of both upstream and downstream sequence, was used to generate transgenic mice. The analysis of mice from six different founder lines demonstrated that this genomic DNA fragment was sufficient to direct human CD14 gene expression in a monocyte-specific manner among hematopoietic cells. Furthermore, the data lead us to a new finding that CD14 is highly expressed in the human liver, a primary organ involved in the acute phase response. These transgenic mice provide a useful model to analyze the biological function of human CD14.     

REP-PCR fragments as biomarkers for differentiating gastroduodenal disease-specific Helicobacter pylori strains

We previously identified four potential putative gastroduodenal disease fragments by using the interspersed repetitive extragenic palindromic DNA sequence based PCR (REP-PCR) technique. We investigated these fragments with regard to their disease specificity. The putative disease-specific REP-PCR fragments were cloned, mapped by restriction enzymes, cross-hybridized, and confirmed by Southern hybridization. The four fragments were also used as probes against REP-PCR amplicons from H. pylori isolates obtained from gastritis (N = 20), duodenal ulcer (N = 30), and gastric cancer patients (N = 30). Three of these fragments (1.4- and 0.76-kb for gastritis; 1.35 kb for duodenal ulcer) were amplified without any discrimination between any disease-specific H. pylori isolates. However, amplification following hybridization with the fourth 0.81-kb fragment was observed only from gastritis (60%) and duodenal ulcer (52%) but with none (0%) of gastric cancer patients. Nucleotide sequence analysis of the 0.81-kb fragment revealed that it was an open reading frame of the hypothetical protein HP0373 matched to the position of 380,966 to 383,068 nucleotides of the H. pylori complete genome sequence. Hence, the REP-PCR sequence was not a extragenic palindromic DNA sequence. The hypothetical protein was also present in all the tested isolates. The REP-PCR fingerprinting technique is useful to differentiate disease-specific H. pylori strains based on the interspersed repetitive extragenic palindromic DNA sequences; however, it may not be useful to identify disease-specific virulence determinant(s) without being confirmed by DNA sequence analysis and functional studies.     

Phosphorylation of p130Cas by angiotensin II is dependent on c-Src, intracellular Ca2+, and protein kinase C

Prorenin is expressed in certain extrarenal tissues, but normally only the kidneys process prorenin to renin and secrete renin into the circulation. Although transgenic animal lines containing the human renin (hREN) structural gene with either 0.9-kb or 3-kb 5'-flanking DNA express the transgene appropriately in renal juxtaglomerular cells and secrete hREN into the circulation, the source of the circulating renin is not known. In the present study, we observed that 13-kb hREN transgenic mice that contain the structural gene and 0.9-kb 5'-flanking DNA express hREN mRNA in many unusual tissues. We also observed that circulating hREN levels in 13-kb hREN mice increased after bilateral nephrectomy. These results suggested that the hREN gene is expressed at inappropriate locations where prorenin might be processed to renin. To determine if more distal sequences flanking the hREN gene might contribute to cell and tissue specificity, we used a 45-kb hREN genomic fragment that contained the structural gene and about 25-kb 5'- and 8-kb 3'-flanking DNA sequences to generate 3 separate transgenic lines that contained the intact transgene sequences. Ribonuclease protection assays revealed a much narrower tissue distribution of hREN expression than in the 13-kb hREN transgenic mice. In each 45-kb hREN line, hREN mRNA was present only in the kidney, adrenal, lung, eye, ovary, and brain. Moreover, 24 hours after nephrectomy, human plasma renin fell to very low levels, indistinguishable from those of nontransgenic littermates, indicating that their circulating hREN is of renal origin. These studies suggest that sequences flanking the structural gene, missing from previous hREN transgenic lines, suppress renin gene expression at inappropriate extrarenal sites where cellular proteases, to which prorenin is not normally exposed, could convert prorenin to renin, resulting in abnormal secretion of renin into the plasma.     

Cloning, expression, and sequencing of a protease gene from Bacteroides forsythus ATCC 43037 in Escherichia coli

We have isolated and characterized an N-benzoyl-Val-Gly-Arg-p-nitroanilide-specific protease gene, designated prtH, from Bacteroides forsythus ATCC 43037. Nucleotide sequencing of the DNA insert from the clone (hereafter referred to as clone FST) revealed that the protease activity corresponded to an open reading frame consisting of 1,272 bp coding for a 47.8-kDa protein. When plasmid pFST was used as a probe in Southern hybridization, Sau3AI-digested chromosomal DNA of B. forsythus ATCC 43037 as well as the chromosomal DNAs of the isolated strains Ta4, TR5, and YG2 showed 0.6- and 0.8-kb hybridizing bands. The cell-free extracts of clone FST showed hemolytic activity on human blood cells. The hydrolytic activity of cell extracts of the pFST clone was inhibited by p-toluenesulfonyl-L-lysine chloromethyl ketone hydrochloride, leupeptin, N-ethylmaleimide, iodoacetic acid, iodoaceteamide, and EDTA.     

Phylogeny of the genus Pistacia as determined from analysis of the chloroplast genome

Classification within the genus Pistacia has been based on leaf morphology and geographical distribution. Molecular genetic tools (PCR amplification followed by restriction analysis of a 3.2-kb region of variable chloroplast DNA, and restriction fragment length polymorphism analysis of the Pistacia cpDNA with tobacco chloroplast DNA probes) provided a new set of variables to study the phylogenetic relationships of 10 Pistacia species. Both parsimony and cluster analyses were used to divide the genus into two major groups. P. vera was determined to be the least derived species. P. weinmannifolia, an Asian species, is most closely related to P. texana and P. mexicana, New World species. These three species share a common origin, suggesting that a common ancestor of P. texana and P. mexicana originated in Asia. P. integerrima and P. chinensis were shown to be distinct whereas the pairs of species were monophyletic within each of two tertiary groups, P. vera:P. khinjuk and P. mexicana:P. texana. An evolutionary trend from large to small nuts and leaves with few, large leaflets to many, small leaflets was supported. The genus Pistacia was shown to have a low chloroplast DNA mutation rate: 0.05-0.16 times that expected of annual plants.     

Focus on children! A model for international development cooperation

An extrachromosomal circular DNA of of approximately 50-kb size was amplified in the hydroxyurea-resistant variant of Leishmania mexicana amazonensis. The amplicon carried the M2 gene of ribonuleotide reductase as part of the gene encoding resistance to hydroxyurea. The amplicon was unstable. It disappeared rapidly as shown in pulse-field gradient electrophoresis gels after reversion of the cells for 20-80 days. This loss of amplified DNA was accompanied by a rapid loss of resistance to hydroxyurea during the same period. The amplicon was not hybridized to specific probes from any of the four regions of DNA amplification previously reported for Leishmania. This region of amplification thus appears to be a new region of DNA amplification in Leishmania.